PROJECT SUMMARY Animals deprived of vitamin A in their diet (VAD) exhibit male sterility. All trans retinoic acid (ATRA), an active metabolite of vitamin A, functions by binding to the nuclear retinoic acid receptors (RARs), of which there are three isoforms ?, ?, and ?. We and others have shown the importance of ATRA signaling via the RAR? receptor in particular during spermatogenesis by gene targeting. Rara-/- mice are viable and healthy but the males are sterile, with defects in spermatogenesis similar to that seen in mice maintained on a VAD diet. Following up on observations of ?testicular toxicity? in rats treated with a pan-RAR antagonist, we have shown that indeed, spermatogenesis is inhibited in male mice after treatment with this orally bioavailable compound, and importantly, that the induced sterility is reversible. Our toxicology studies did not reveal any detectable side effects and normal progeny were sired after restoration of fertility. This suggests that antagonizing RAR signaling and RAR? in particular, has potential as a novel and non-steroidal hormone-based approach to male contraception. The objective in this application is to identify retinoids with that selectively antagonize RAR ? and to evaluate their therapeutic efficacy and reversibility. In Specific Aim 1, we propose to design, synthesize, and evaluate the in vitro activity, binding, and ADMET properties of RAR?-selective antagonists. Binding studies will utilize isothermal titration calorimetry, differential scanning fluorimetry, surface plasmon resonance, microscale thermophoresis, and X-ray crystallography; activity studies will use a luciferase reporter assay; and pharmacological profiles will be assessed. Specific Aim 2 will then assess the in vivo effects of the most promising RAR?-selective antagonists on spermatogenesis. After the initial morphological analysis, we will conduct fertility studies and develop dosing regimens for the most promising candidate molecules to determine the lowest doses and longest dosing periods at which efficient induction of sterility with complete reversibility could be achieved. Specific Aim 3 will then move to testing the lead candidate drug in a non-human primate, the common marmoset, model in a small scale trial to set the stage for future pre-clinical trials. Finally, we have observed in both our genetic and pharmacological models that one of the physiological processes in spermatogenesis that is extremely sensitive to altered retinoid signaling is spermiation. In Specific Aim 4, we therefore propose to determine both the quantitative global proteome and the quantitative phospho-proteome in particular during spermiation using dissected segments of testicular tubules and analysis by mass spectrometry.